Differential Desensitization Observed at Multiple Effectors of Somatic μ-Opioid Receptors Underlies Sustained Agonist-Mediated Inhibition of Proopiomelanocortin Neuron Activity

Abstract

Activation of somatic μ-opioid receptors (MORs) in hypothalamic proopiomelanocortin (POMC) neurons leads to the activation of G-protein-coupled inward rectifier potassium (GIRK) channels and hyperpolarization, but in response to continued signaling MORs undergo acute desensitization resulting in robust reduction in the peak GIRK current after minutes of agonist exposure. We hypothesized that the attenuation of the GIRK current would lead to a recovery of neuronal excitability whereby desensitization of the receptor would lead to a new steady state of POMC neuron activity reflecting the sustained GIRK current observed after the initial decline from peak with continued agonist exposure. However, electrophysiologic recordings and GCaMP6f Ca²⁺ imaging in POMC neurons in mouse brain slices indicate that maximal inhibition of cellular activity by these measures can be maintained after the GIRK current declines. Blockade of the GIRK current by Ba²⁺ or Tertiapin-Q did not disrupt the sustained inhibition of Ca²⁺ transients in the continued presence of agonist, indicating the activation of an effector other than GIRK channels. Use of an irreversible MOR antagonist and Furchgott analysis revealed a low receptor reserve for the activation of GIRK channels but a >90% receptor reserve for the inhibition of Ca²⁺ events. Altogether, the data show that somatodendritic MORs in POMC neurons inhibit neuronal activity through at least two effectors with distinct levels of receptor reserve and that differentially reflect receptor desensitization. Thus, in POMC cells, the decline in the GIRK current during prolonged MOR agonist exposure does not reflect an increase in cellular activity as expected.SIGNIFICANCE STATEMENT Desensitization of the μ-opioid receptor (MOR) is thought to underlie the development of cellular tolerance to opiate therapy. The present studies focused on MOR desensitization in hypothalamic proopiomelanocortin (POMC) neurons as these neurons produce the endogenous opioid β-endorphin and are heavily regulated by opioids. Prolonged activation of somatic MORs in POMC neurons robustly inhibited action potential firing and Ca²⁺ activity despite desensitization of the MOR and reduced activation of a potassium current over the same time course. The data show that somatic MORs in POMC neurons couple to multiple effectors that have differential sensitivity to desensitization of the receptor. Thus, in these cells, the cellular consequence of MOR desensitization cannot be defined by the activity of a single effector system.

Keywords: GIRK; action potential; calcium imaging; electrophysiology; hypothalamus; receptor reserve.

Figure 1.

Sustained inhibition of AP firing and Ca²⁺ activity by MOR activation. A, Voltage-clamp recording from a POMC neuron showing attenuation of the GIRK current elicited by ME (10 μm) during a 10 min application. The residual GIRK current is eliminated the MOR selective antagonist CTAP (500 nm). Dotted lines are overlaid to highlight the baseline current (bottom dashed line), the peak GIRK current (top dashed line), and the steady-state GIRK current (middle dashed line) following desensitization. B, Summary data showing the attenuation of the GIRK current during prolonged ME exposure (n = 6 neurons, 4 animals). C, Extracellular loose-patch recording of a Pomc-Cre neuron expressing GCaMP6f during a 10 min ME (10 μm) application with subsequent reversal by CTAP (500 nm). Top trace, Action currents recorded in loose patch, with the bottom plot showing AP frequency over time. D, Summary data of the sustained inhibition of AP frequency by ME (10 μm; n = 6 neurons, 6 animals) over a 10 min application. E, Optical recording of the same neuron as in C. Top trace is the F/F₀ normalized GCaMP6f fluorescence intensity with measured Ca²⁺ event frequency plotted below. In this cell, ME (10 μm) completely eliminated both AP firing and resolvable Ca²⁺ events. F, Summary data for the inhibition of Ca²⁺ events by ME (n = 29 neurons, 5 slices, 3 animals).

Figure 2.

Blockade of GIRK current by barium fails to prevent the inhibition of Ca²⁺ events by MORs or uncover receptor desensitization. A, GCaMP6f fluorescence recording (F/F₀) where Ba²⁺ (100 μm) was applied before ME (10 μm) showing a complete cessation of resolvable Ca²⁺ events. B, Summary plot of the inhibition of GCaMP6f events by ME (10 μm) in the presence of 100 μm Ba²⁺ (n = 22 neurons, 3 slices, 3 animals). C, Experiment as performed in A, but with [Ba²⁺] increased to 400 μm. D, Summary plot of inhibition of GCaMP6f events by ME (10 μm) in the presence of 400 μm Ba²⁺ (n = 18 neurons, 3 slices, 3 animals). E, Experiment as performed in A, but with [Ba²⁺] increased to 1 mm. F, Summary plot of inhibition of GCaMP6f events by ME (10 μm) in the presence of 1 mm Ba²⁺ (n = 12 neurons, 2 slices, 2 animals). Only the time point at minute 16 is significantly different than that at minute 10. G, Whole-cell voltage-clamp recording of the GIRK current elicited by ME (10 μm) in the presence of Ba²⁺ (100 μm) illustrating the blunted GIRK current compared with Figure 1A. H, Summary of the magnitude of GIRK currents from POMC neurons elicited by ME (10 μm) alone (n = 11 neurons, 7 animals, black circles), which is significantly reduced in the presence of 100 μm Ba²⁺ (n = 8 neurons, 6 animals, black squares). I, Dose–response curves for the inhibition of GCaMP6f events by ME. Under control conditions (solid black line) the EC₅₀ value was 130 nm, and blockade of AMPA and GABA_A receptors (dotted blue line, DNQX and picrotoxin, respectively) did not significantly alter the EC₅₀ value (90 nm). In the presence of Ba²⁺ (100 μm), there was a significant twofold rightward shift of the EC₅₀ value (270 nm).

Figure 3.

Blockade of the ME-induced GIRK current by the GIRK selective blocker Tertiapin-Q fails to prevent the inhibition of Ca²⁺ events by ME. A, Whole-cell voltage-clamp recording from a POMC neuron. A brief (90 s) pulse of ME (10 μm) was used to elicit a GIRK current just before the addition of the GIRK selective blocker Tertiapin-Q (500 nm). Following blockade with Tertiapin-Q, a 3 min pulse of ME was used to determine the amplitude of the residual GIRK current. B, Dot plot showing the amplitude of the GIRK current elicited by ME (10 μm) before and after the addition of Tertiapin-Q (500 nm; n = 8 cells, 5 animals). C, Slice average (mean ± SEM) plotting the frequency of GCaMP6f events in response to ME (10 μm) before and after the addition of Tertiapin-Q (500 nm) to block GIRK channels. D, Summary data for the inhibition of Ca²⁺ events by ME (10 μm) in the presence of 500 nm Tertiapin-Q (n = 60 neurons, 9 slices, 3 animals).

Figure 4.

Desensitization of the MOR reduces the inhibition of Ca²⁺ events by an EC₅₀ concentration of ME. A, Normalized (F/F₀) fluorescent GCaMP6f recording illustrating the protocol used to detect MOR desensitization. In the presence of Ba²⁺ (100 μm), to minimize the GIRK current, a near-EC₅₀ concentration of ME (300 nm) was briefly applied then washed out. Then a 10 min ME (10 μm) application was used to induce MOR desensitization. After washout, a second application of 300 nm ME was used to assess the level of desensitization. B, Slice average (mean ± SEM) for the experiment in A. C, Plot of the percentage inhibition of Ca²⁺ events before (open circles) and after MOR desensitization (open squares). Each point is an individual neuron, and the horizontal bars are the mean ± SEM. The mean inhibition of Ca²⁺ events for the first application of 300 nm ME was significantly greater than the mean inhibition for the second application of 300 nm ME (black asterisk; n = 30, neurons, 6 slices, 4 animals). D, Same data as in C paired to show the downward trend between the first and second applications of 300 nm ME.

Figure 5.

Blockade of the ME-induced GIRK current by the irreversible antagonist β-CNA. A, Whole-cell voltage-clamp recording from a POMC neuron treated for 2 min with β-CNA (50 nm) before a 2 min application of ME (10 μm) used to assess GIRK current amplitude. After washout of ME, nociceptin (200 nm) was used to activate the GIRK current through the nociceptin receptor. B, Plot of the amplitude of the GIRK current from individual neurons. Horizontal bars are the mean ± SEM. The mean amplitude of the GIRK current induced by ME (10 μm, open circles; n = 11 neurons, 7 animals) was significantly reduced by β-CNA pretreatment (50 nm, open squares; n = 9 neurons, 6 animals). The mean amplitude of the GIRK current elicited by nociceptin (200 nm, open triangles; n = 5 neurons, 4 animals) after β-CNA (50 nm) pretreatment was similar to the current elicited by ME in the absence of β-CNA.

Figure 6.

Determination of MOR reserve for the inhibition of Ca²⁺ events by ME. A, Slice average (mean ± SEM) plotting the frequency of GCaMP6f events in response to increasing concentrations of ME (300 nm, 1 μm, and 10 μm) following a 2 min β-CNA (50 nm) pretreatment. GABA_A and AMPA receptor blockers (DNQX and picrotoxin, respectively) were included throughout the recording to occlude the potential effects of presynaptic MORs. B, Dose–response curves for the inhibition of Ca²⁺ events by ME under control conditions or after pretreatment with 50 or 100 nm β-CNA. Treatment with 50 nm β-CNA caused a significant rightward shift of the EC₅₀ concentration (control, 95 nm; 50 nm β-CNA, 1260 nm), without a concomitant decrease in the maximal inhibition, suggesting the presence of a large receptor reserve. Treatment with 100 nm β-CNA did not significantly shift the EC₅₀ concentration (1410 nm) compared with 50 nm, but there was a decrease in the maximal inhibition to 80%. C, Double reciprocal plot of the concentration of agonist required for equally effective points on the dose–response curve between control (y-axis) and β-CNA treatments (x-axis) as used for the Furchgott (1966) analysis, to calculate q, the proportion of receptors still functional following β-CNA treatment, from the slope of the linear fits.